PROJECT SUMMARY Neuroblastoma (NB) remains one of the deadliest childhood cancers. Our long term goal is to improve the outcome of children diagnosed with NB by characterizing the germline and somatic events driving tumorigenesis so that rational, evidence-based therapies can be developed. Our objective here is to perform a primary integrative analysis of the Gabriella Miller Kids First (GMKF) X01 HL136997 NB cohort to identify both inherited and acquired genetic alterations and mutational signatures in NB that may be exploitable for risk prediction and/or therapeutic intervention. Through this research program, we have sequenced 2,277 individual samples and generated 2,655 DNA/RNA sequences. These data include robustly-annotated patient-parent triads/dyads (n=593) and matched tumor DNA (n=366) and RNA (n=228) sequencing. Our central hypothesis is that both inherited and acquired coding and non-coding mutations influence NB initiation and progression. Here, we will test our hypothesis and accomplish our objective in two specific aims: Aim 1) Identify and assess heritability of pathogenic coding and non-coding variants in NB triads and dyads. Rare coding and non-coding single nucleotide variants (SNVs), indels, and structural variants (SVs) will be investigated through a well-developed computational pipeline which we will now extend to the study of triad and dyad data to determine heritability. Pathogenicity will be assessed using multiple algorithms along with the integration of non-coding RNA and epigenetic data from in-house ChIP-seq, ATAC-seq and Capture C data together with public (e.g. ENCODE, Epigenomic Roadmap) to evaluate non-coding variation. Gene-based gTDT, burden and enrichment testing will be performed. Findings will be further evaluated in our parallel pan-childhood cancer germline study of 4,573 children spanning major cancer subtypes and also in children with NB and structural birth defects included in X01 HL140554. Aim 2) Perform integrative tumor-normal analyses to elucidate functional relevance of genetic risk factors. Tumor DNA and RNA sequencing data will be processed through our pipelines designed to evaluate SNV, indels, SVs, and copy number in DNA and account for the study of isoforms, fusions and other novel transcripts in RNA. Next, we will perform an integrative in silico evaluation of recurrent germline events using matched tumor DNA and RNA sequencing and extending to the 1,180 paired germline-tumor samples profiled in the NCI-TARGET project. Mutational signatures will be evaluated, and epistatic interactions assessed. By integrating the NB X01 cohort with extant NB (epi)genomics data, we expect to catalyze our understanding of the genetic basis of NB, with insights here being applicable to the genetic basis of other childhood conditions. Moreover, we will address, for the first time, whether NB genetic risk factors are inherited or acquired de novo. Completion of this project will have a sustained and positive impact on the field by identifying clinically actionable genetic alterations in this important childhood cancer.